This invention relates to newly identified peptide-based gemini surfactant compounds, to the use of such compounds and to their production. The invention also relates to the use of the peptide-based gemini compounds to facilitate the transfer of compounds into cells for drug delivery.
Surfactants are substances that markedly affect the surface properties of a liquid, even at low concentrations. For example surfactants will significantly reduce surface tension when dissolved in water or aqueous solutions and will reduce interfacial tension between two liquids or a liquid and a solid. This property of surfactant molecules has been widely exploited in industry, particularly in the detergent and oil industries. In the 1970s a new class of surfactant molecule was reported, characterised by two hydrophobic chains with polar heads which are linked by a hydrophobic bridge (Deinega,Y et al., Kolloidn. Zh. 36, 649, 1974). These molecules, which have been termed xe2x80x9cgeminixe2x80x9d (Menger, FM and Littau,CA, J.Am.Chem.Soc. 113, 1451, 1991), have very desirable properties over their monomeric equivalents. For example they are highly effective in reducing interfacial tension between oil and water based liquids and have a very low critical micelle concentration.
Cationic surfactants have been used inter alia for the transfection of polynucleotides into cells in culture, and there are examples of such agents available commercially to scientists involved in genetic technologies (for example the reagent Tfx(trademark)-50 for the transfection of eukaryotic cells available from Promega Corp. WI, USA).
The efficient delivery of DNA to cells in vivo, either for gene therapy or for antisense therapy, has been a major goal for some years. Much attention has concentrated on the use of viruses as delivery vehicles, for example adenoviruses for epithelial cells in the respiratory tract with a view to corrective gene therapy for cystic fibrosis (CF). However, despite some evidence of successful gene transfer in CF patients, the adenovirus route remains problematic due to inflammatory side-effects and limited transient expression of the transferred gene. Several alternative methods for in vivo gene delivery have been investigated, including studies using cationic surfactants. Gao,X et al. (1995) Gene Ther. 2, 710-722 demonstrated the feasibility of this approach with a normal human gene for CF transmembrane conductance regulator (CFTR) into the respiratory epithelium of CF mice using amine carrying cationic lipids. This group followed up with a liposomal CF gene therapy trial which, although only partially successful, demonstrated the potential for this approach in humans (Caplen, N J. et al., Nature Medicine, 1, 39-46, 1995). More recently other groups have investigated the potential of other cationic lipids for gene delivery, for example cholesterol derivatives (Oudrhiri,N et al. Proc.Natl.Acad.Sci. 94, 1651-1656, 1997). This limited study demonstrated the ability of these cholesterol based compounds to facilitate the transfer of genes into epithelial cells both in vitro and in vivo, thereby lending support to the validity of this general approach.
These studies, and others, show that in this new field of research there is a continuing need to develop novel low-toxicity surfactant molecules to facilitate the effective transfer of polynucleotides into cells both in vitro for transfection in cell-based experimentation and in vivo for gene therapy and antisense treatments. The present invention seeks to overcome the difficulties exhibited by existing compounds.
The invention relates to the peptide-based gemini compounds comprising two linked chains: 
each chain having:
(1) a positively charged hydrophilic head, Q1 or Q2, formed from one or more amino acids and/or amines;
(2) a central portion, P1 or P2, having a polypeptide backbone; and
(3) a hydrophobic tail, R1 or R2; the central sections of each chain being linked together by bridge Y through residues in P1 and P2.
Preferably the central portion is made up of two or three amino acids, Pa (optional), Pb and Pc, in which:
Pa is a D- or L- amino acid, preferably hydrophilic, such as threonine or serine,
Pb is preferably D- or L- cysteine, serine or threonine, and
Pc is preferably D- or L- serine or threonine and is linked to R1 or R2.
Preferred compounds of the present invention include compounds of the formula (I): 
where:
A1 and A5 which may be the same or different, is a positively charged group formed from one or more amino acids or amines joined together in a linear or branched manner and preferably bonded by an amide (CONH) bond;
A2/A6CH(NH)CO, which may be the same or different, is derived from an amino acid, preferably serine;
p and q, which may be the same or different, is 0 or 1;
X1/X2CH2CH(NH)CO, which may be the same or different, is derived from cysteine (X1/X2xe2x95x90S), serine or threonine (X1/X2xe2x95x90O);
A4/A8CH(NH)CO, which may be the same or different, is derived from serine or threonine;
Y is a linker group, preferably (CH2)m where m is an integer from 1 to 6, most preferably 2, and may be a disulphide bond when X1 and X2 is each S;
R1 and R2 are C(10-20) saturated or unsaturated alkyl groups, and
W and Z are NH, O, CH2 or S; or
a salt, preferably a pharmaceutically acceptable salt thereof.
Preferably, the compound is symmmetrical, that is A1 and A5 are the same, A2 and A6 are the same, A4 and A8 are the same, R1 and R2 are the same, and W and Z are the same.
Representative examples of A1/A5 include D- or L-amino acids selected from arginine, lysine, ornithine and histidine, preferably lysine, or amines such as spermine and spermidine. Up to seven amino acids and/or amines may be linked in a linear or branched chain. Prefered examples include groups having two or three lysines or ornithines or a combination of lysine, ornithine, arginine and histidine, for instance:
COCH(NHR)(CH2)4NHCO(NH2)(CH2)4NH2
or
COCH(NHR)(CH2)3NHCO(NH2)(CH2)3NH2
or
COCH(NHR)(CH2)4NHCO(NH2)(CH2)3NH2
in which R is H or NHCO(NH2)(CH2)4NH2 or NHCO(NH2)(CH2)3NH2 
Preferably, xe2x80x94X1xe2x80x94Yxe2x80x94X2xe2x80x94 is xe2x80x94SCH2CH2Sxe2x80x94 or xe2x80x94OCH2CH2Oxe2x80x94
Preferably, R1 and R2 is each a C12-C20 alkyl group, for instance C12.
Preferably, W and Z is NH, thereby forming a further amide (CONH) bond.
Compounds of the present invention may be prepared from readily available starting materials using synthetic peptide chemistry well known to the skilled person. For prefered compounds of the present invention a useful intermediate is the compound: 
which is synthesised in a multi-stage process beginning, for instance, with the construction of the di-cysteine part and subsequently building up the hydrophilic head by attaching a serine moiety at the carboxyl group of each cysteine moiety, using standard peptide chemistry, and then attaching the hydrocarbon chains to the carboxyl group of the serine moiety using a standard amide forming reaction well known to those skilled in the art. This intermediate can then be taken through to compounds of formula (I) by further reaction at the nitrogens of the cysteine residues.
Another aspect of the invention relates to methods for using the peptide-based gemini compounds. Such uses include facilitating the transfer of oligonucleotides and polynucleotides into cells for antisense, gene therapy and genetic immunisation (for the generation of antibodies) in whole organisms. Other uses include employing the compounds of the invention to facilitate the transfection of polynucleotides into cells in culture when such transfer is required, in, for example, gene expression studies and antisense control experiments among others. The polynucleotides can be mixed with the compounds, added to the cells and incubated to allow polynucleotide uptake. After further incubation the cells can be assayed for the phenotypic trait afforded by the transfected DNA, or the levels of mRNA expressed from said DNA can be determined by Northern blotting or by using PCR-based quantitation methods for example the Taqman(copyright) method (Perkin Elmer, Connecticut, USA). Compounds of the invention offer a significant improvement, typically between 3 and 6 fold, in the efficiency of cellular uptake of DNA in cells in culture, compared with compounds in the previous art. In the transfection protocol, the gemini compound may be used in combination with one or more supplements to increase the efficiency of transfection. Such supplements may be selected from, for example:
(i) a neutral carrier, for example dioleyl phosphatidylethanolamine (DOPE) (Farhood, H., et al (1985) Biochim. Biophys. Acta 1235 289);
(ii) a complexing reagent, for example the commercially available PLUS reagent (Life Technologies Inc. Maryland, USA) or peptides, such as polylysine or polyomithine peptides or peptides comprising primarily, but not exclusively, basic amino acids such as lysine, ornithine and/or arginine. The list above is not intended to be exhaustive and other supplements that increase the efficiency of transfection are taken to fall within the scope of the invention.
In still another aspect, the invention relates to the transfer of genetic material in gene therapy using the compounds of the invention.
Yet another aspect of the invention relates to methods to effect the delivery of non-nucleotide based drug compounds into cells in vitro and in vivo using the compounds of the invention.
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
xe2x80x9cAmino acidxe2x80x9d refers to dipolar ions (zwitterions) of the form +H3NCH(R)CO2xe2x88x92. They are differentiated by the nature of the group R, and when R is different from hydrogen can also be asymmetric, forming D and L families. There are 20 naturally occurring amino acids where the R group can be, for example, non-polar (e.g. alanine, leucine, phenylalanine) or polar (e.g. glutamic acid, histidine, arginine and lysine). In the case of un-natural amino acids R can be any other group which is not found in the amino acids found in nature.
xe2x80x9cPolynucleotidexe2x80x9d generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. xe2x80x9cPolynucleotidesxe2x80x9d include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, xe2x80x9cpolynucleotidexe2x80x9d refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. xe2x80x9cModifiedxe2x80x9d bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications have been made to DNA and RNA; thus, xe2x80x9cpolynucleotidexe2x80x9d embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. xe2x80x9cPolynucleotidexe2x80x9d also embraces relatively short polynucleotides, often referred to as oligonucleotides.
xe2x80x9cTransfectionxe2x80x9d refers to the introduction of polynucleotides into cells in culture using methods involving the modification of the cell membrane either by chemical or physical means. Such methods are described in, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The polynucleotides may be linear or circular, single-stranded or double-stranded and may include elements controlling replication of the polynucleotide or expression of homologous or heterologous genes which may comprise part of the polynucleotide.